Cytotoxic Flavonoid Glycosides from Rapistrum rugosum L.

Five flavonoid glycosides were isolated from the n-butanol soluble fraction of the ethanolic extract of Rapistrum rugosum and their structures were assigned from 1H- and 13C-NMR spectra (DEPT) with 2D NMR as quercetin-3-O-α-L-rhamnopyranoside (1), quercetin-3-O- β-D-xyloside (2), quercetin, 3-O-α-L-arabinopyranoside,7-O-α-L-rhamnopyranoside (3), kaempferol 3-O-α-L-arabinopyranoside, 7-O-α-L-rhamnopyranoside (4) and rutin (5). The SRB cytotoxic assay was used to investigate the antitumor activities of n-butanol extract, compound 3 and its hexaacetate 3a, for the first time. Compounds 3 and 3a showed cytotoxic activity against the human cancer cell line, namely, HepG2 (hepatocellular carcinoma cell line) with IC50 (concentration of compound required to reduce cell survival by 50%) 0.86 μg/mL and 3.50 μg/mL, respectively. These results proved that compound 3, the major flavonoid of the n-butanol soluble fraction, has significant cytotoxic activity compared with the standard antitumor drug doxorubicin (0.60 μg/mL).


Introduction
Rapistrum rugosum L. commonly known as turnip weed, wild turnip or bastard cabbage, belongs to the family of Cruciferae. This family comprises about 390 genera and is represented in Saudi Arabia by 49 genera. R. rugosum is native to North Africa, Europe, the Middle East and Pakistan and is the only Rapistrum species in Saudi Arabia (1). The leaves of R. rugosum are externally applied to the heal legs furuncles in Italy (2). R. rugosum is boiled and used for culinary purposes as one of the most popular, wild food plants in Sicily (3).
The high demand of the innovative lead structures to develop the novel drugs for the treatment of cancer and other menacing diseases drove us to study the cytotoxic activity of the ethanolic plant extract. The ethanolic extract of R. rugosum showed cytotoxic activity, on further biological screening of all fractions, the n-BuOH soluble fraction revealed a strong cytotoxic activity. This prompted us to carry out the phytochemical study and try to isolate the constituents of the n-BuOH soluble fraction of R. rugosum. Our goal was also to try and investigate the effect of acetylation on the cytotoxic activity of the major isolated compound.
Fraction C (1.5 g) was a binary mixture which was separated on a cellulose column with 80% MeOH/H 2 O to afford 1 (15 mg) and 2 (12 mg).

Acid Hydrolysis of compound 3
Compound 3 (5 mg) in MeOH (5 mL) containing 1N HCl (5 mL) was refluxed for 4 h, concentrated under reduced pressure and diluted with H 2 O (10 mL). It was extracted with EtOAc and the residue recovered from the organic phase yielded quercetin as an aglycone. The remaining aqueous solutions were evaporated to dryness, resolved in MeOH and subjected to TLC analysis (eluent: EtOAc-MeOH-H 2 O-HOAc, 6:2:1:1). The chromatogram was sprayed with aniline hydrogen phthalate followed by heating at 100°C. The sugars were identified after the comparison through authentic standards.

Cytotoxicity assay In-vitro SRB cytotoxic assay against Human liver cancer cell line (HepG2 cells)
Potential cytotoxicity of n-butanol extract, compounds 3 and 3a were tested using the method of Skehan and Storeng (4). The sensitivity of the human tumor cell lines to thymoquinone was determined through the SRB assay. SRB is a bright pink aminoxanthrene dye with two sulphonic groups. It is a protein stain that binds to the amino groups of intracellular proteins under mildly acidic conditions to provide a sensitive index of cellular protein content. Hepatocellular cell line (HepG2) was obtained frozen in liquid nitrogen at -180°C from the American Type and 13 C-NMR, respectively. The chemical shift values are reported in ppm (δ) unit and the coupling constants (J) are in Hz. The column chromatography was carried out on various adsorbents including silica gel 230-400 mesh, cellulose and sephadex LH-20 (E. Merck, Darmstadt, Germany). FAB and HRFABMS (neg. mode, matrix: glycerol) on JEOL JMS-HX110, mass spectrometer and Thin layer chromatography (TLC) were performed on precoated silica gel F 254 plates (E. Merck, Darmstadt, Germany), silica gel RP-18 F 254 and cellulose. The detection was done at 254 nm by spraying with ceric sulphate and AlCl 3 reagents.

Plant material
The aerial parts of R. rugosum (1.00 Kg) were collected from Riyadh (Saudi Arabia) in
Fraction A (3 g) was chromatographed on a Sephadex LH-20 column using n-BuOH/iso-pr. OH/H 2 O (BIW, 4:1:5, organic layer) for elution to afford 4 sub-fractions (I-IV). Sub-fraction II contained only one spot and it was then purified on a Sephadex LH-20 column with MeOH (eluent) to give compound 3 (75 mg). Subfraction III was chromatographed on a cellulose column with 80% MeOH/H 2 O as eluent and the collected fraction was further purified on Sephadex column with EtOH to yield compound 4 (15 mg).

Results and Discussion
The mixture of flavonoid glycosides obtained from the n-BuOH fraction of the ethanolic extract of R. rugosum was subjected to a series of column chromatographic separations to isolate compounds 1-5, namely; quercetin-3-O-α-L-rhamnopyranoside (1)    The major compound 3 was derivatized into its hexaacetate derivative (3a) and analyzed through HRFABMS and NMR. The major flavonoid diglycoside (3) and its hexaacetate (3a) were screened for cytotoxicity against the human cancer cell line, namely, HepG2 (hepatocellular carcinoma cell line). From the results shown in Table 1 and Figures 1 and 2, it could be seen that compound 3 shows a significant cytotoxic activity against the liver carcinoma cell line (IC 50 = 0.86 µg/mL), while the acetylated compound 3a shows a lower cytotoxic activity (IC 50 = 3.50 µg/mL) compared to the standard drug doxorubicin (IC 50 = 0.60 µg/mL).
Quercetin Hz, H-6′). In the aliphatic region, an anomeric proton signal at δ 5.30 (d, J = 4.5 Hz), together with two oxymethylene protons observed at δ 3.24 (m) and δ 3.62 (m) were indicative to the presence of α-L-arabinopyranoside moiety. The second anomeric proton was observed at δ 5.56 as (brs). The 1 H NMR spectrum further showed signals of oxymethine protons in the range of δ 3.76-3.10 and the methyl protons resonated at δ 1.12 (d, J = 5.5 Hz) which was characteristic for α-L-rhamnopyranoside moiety.
The 13 C NMR and DEPT spectra showed twenty-six signals comprising one methyl, one methylene, fourteen methine and ten quaternary carbons. The signals at δ 156.7, 133.8, 177.6 and 105.5 were typical of C-2, C-3, C-4 and C-10 of a flavonol moiety. The signals of two anomeric carbons of the sugar moieties appeared at δ 101.2 and 99.4. Assignment of all 1 H and 13 C resonances was proved through their comparison with the reported data in the literature (6-13).  L-rhamnose and it was confirmed through the TLC of sugars with their standards.
In addition, the major flavonoid diglycoside (3) and its acetylated form (3a) were screened for cytotoxicity against the human cancer cell line, namely, HepG2 (hepatocellular carcinoma cell line).
Previous studies, however, proved the antitumor activity of flavonoids and even aimed at elucidating the structure-activity relationships in order to develop new anticancer drugs (14). This is the first report for the cytotoxic activity of these compounds. This finding may help to show the structural requirements implicated in the anticancer activity of flavonoids, with the goal of rationalizing their development as antitumor agents.